Carswell et al. found that a serum sample taken from a mouse stimulated with Bacillus Calmette-Guerin (BCG) and then given an endotoxin contains a substance which bleeds and necrotizes a solid tumor caused by a transplanted Meth A sarcoma; and named this substance tumor necrosis factor (abbreviated as "TNF") (E. A. Carswell et al., Proc. Natl. Acad. Sci., U. S. A. 72, 3666 (1975)). TNF is found in many animals such as mice, rabbits and humans. Since it acts specifically on tumor cells of any species, it is expected to be used as an antitumor agent.
Recently, Pennica et al. disclosed the primary structure of a human TNF protein by cloning cDNA of human TNF, and reported on the expression of the human TNF gene in Escherichia coli (D. Pennica et al.: Nature, 312, 724 (1984)). Later, Shirai et al. (T. Shirai et al.: Nature, 313, 803 (1985)), Somura et al. [Somura et al.: Cancer and Chemotherapy, 12, 160 (1985)), Wang et al. (A. M. Wang et al.: Science, 228, 149 (1985)), and Marmenout et al. (A. Marmenout et al.: Eur. J. Biochem., 152, 515 (1985)) reported the expression of human TNF genes in E. coli.
Thus, large quantities of pure human TNF proteins have become available by using the genetic manipulation technology, and physiological activities of TNF other than the antitumor activity have been elucidated in more detail. For example, it was suggested that cachectin, a substance which is one cause of inducing cachexia in patients in the terminal stage of cancer or patients with serious infections, is very similar to TNF (B. Beulter et al.: "Nature", 316, 552 (1985)), and since cachectin has lipoprotein lipase inhibitory activity, the administration of TNF increases the amount of triglycerides in the blood, and may possibly induce side-effects such as hyperlipemia. Elsewhere, the influence of TNF on vascular endothelial cells (J. R. Gamble et al.: J. Exp. Med., 162, 2163 (1985)), and its bone absorbing action (D. R. Beltolini et al.: "Nature", 319, 516 (1986)) have been reported.
On the other hand, the recent advance in genetic manipulation technology has enabled gene recombination to substitute an amino acid in a useful protein by another amino acid, to add an amino acid or to delete an amino acid from it. A number of research works have been conducted for modifying a naturally occurring protein and creating proteins which meet a specific purpose.
On the other hand, with regard to the modification of human TNF proteins, a number of research works have been made; there have been reported substitution of either or both of amino acid residues of CYs.sup.69 and Cys.sup.101 by other amino acid residues in the amino acid sequence of human TNF proteins shown in FIG. 1 ( see PCT Application Laid-Open Specification W086/04606 and Japanese Laid-Open Patent Publication No. 263199/1987), substitution of Gly.sup.122 by another amino acid residue (Japanese Laid-Open Patent Publication Nos. 263199/1987 and 93799/1988), and substitution of Ala.sup.18 by another amino acid (Japanese Laid-open Patent Publication No. 87996/1988). Moreover, regarding the deletion of amino acids on the amino-terminus side, it has been reported that TNF in which amino acids Nos. 1 to 6 are deleted has antitumor activity (Japanese Laid-Open Patent Publication No. 50923/1986), that TNF in which amino acids Nos. 1 to 7 are deleted has antitumor activity (Japanese Patent Application No. 90087/1986), that TNF in which amino acids Nos. 1 to 10 are deleted has antitumor activity and the specific activity is highest in TNF's in which animo acids Nos. 1 to 6, 1 to 7 and 1 to 8 are deleted (PCT Application Laid-Open Specification W086/02381), that TNF in which amino acid Nos. 1 to 10 are deleted has antitumor activity (Japanese Laid-Open Patent Publication No. 272991/1987), that TNF in which amino acids Nos. 1 to 11 are deleted has cytotoxicity (Japanese Laid-Open Patent Publication No. 32486/1988), and that regarding TNF in which amino acids Nos. 1 to 7 are deleted, TNF with -Pro-Ser-Asp- amino acids Nos. 8 to 10 substituted by -Arg-Lys-Arg amino acids are much increased in the specific activity (Japanese Laid-Open Patent Publication No. 188396/1988).
Accordingly, the present inventors have made research works to produce novel modified human TNF proteins with a view to improving specific activity and stability, broadening a zone of a reaction spectrum and reducing side effects, and consequently arrived at the present invention.